Article Figures & Data

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ALA sensitizes human skin to blue light illumination. In healthy human volunteers, ALA was injected into the epidermal layer of the skin. After a period of 3 h, the skin was illuminated with a 405 nm laser. Subjects rated the painful sensation on a numerical scale (1–100) every 5 s; the illumination was terminated after 60 s or when reaching a rating of 50. A, Time course of individual pain ratings induced by blue light after ALA 10 mm or control solution. The trials of the same subject are indicated by the same symbol. Recovery was recorded for 20 s after the end of the light stimulation. B, The averaged pain rating of 7 volunteers shows a concentration-dependent increase in the pain rating after ALA compared with control. For trials terminated earlier, a rating of 50 was extrapolated for all subsequent time points. C, ALA pretreatment photosensitized, which manifests as an increased area under the curve (AUC) of the rating, an increased maximum rating reported in a trial, and a decreased trial duration. *p < 0.05.

Exposure to UV light induces membrane currents in hTRPA1- but not in hTRPV1-expressing cells. A, Example of a whole-cell patch-clamp recording from a hTRPA1-expressing HEK293t cell stimulated by UV light (<410 nm) and carvacrol (Carv., 100 μm). Following UV stimulation, the TRPA1 antagonist HC-030031 (HC, 10 μm) was applied, leading to a complete inhibition of the current. B, Example of a whole-cell patch-clamp recording from a hTRPV1-expressing HEK293t cell stimulated by UV light (blue bar) and capsaicin (Caps., 1 μm). Illumination with UV light for 30 s did not evoke a TRPV1-mediated current in this cell. A, B, Plot of currents at −80 mV (squares) and 80 mV (circles) extracted from voltage ramps. C, D, Ramp currents recorded at the times indicated with the corresponding color in A and B.

Pretreatment with ALA dramatically increases the photosensitivity of hTRPA1 and hTRPV1. HEK293t cells expressing hTRPA1 (A), hTRPV1 (B), and untransfected control cells were preincubated with ALA (300 μm) for 3 h in the dark at 37°C. The 340/380 nm illumination (10 ms each, 1 Hz) increased intracellular calcium in cells expressing hTRPA1 and hTRPV1 but not in control HEK293t cells. hTRPA1 (C) and hTRPV1 (D) expressing cells preincubated with ALA (300 μm, 3 h) were imaged in the presence or absence of extracellular calcium. The calcium increase by the 340/380 nm illumination was abolished in calcium-free conditions, but large calcium transients occurred upon returning to a calcium-containing extracellular solution. Simply removing extracellular calcium in untreated hTRPA1 or hTRPV1-expressing cells only produced small calcium transients upon returning to a calcium-containing extracellular solution. E, F, Cells were preincubated with ALA (300 μm, 3 h) and imaged in the presence or absence of the TRPA1 antagonist HC-030031 (10 μm, HC, E) and TRPV1 antagonist BCTC (1 μm, F). Calcium transients upon removal of HC-030031 were larger in ALA-pretreated compared with untreated hTRPA1-expressing cells. In contrast, washout of BCTC did not result in calcium transients in hTRPV1-expressing cells. Data are mean ± SEM. G, Example of a whole-cell patch-clamp recording from an hTRPA1-expressing cell pretreated with ALA (300 μm, 3 h). Stimulation by blue light (460–490 nm) induced a current that was inhibited by HC-030031 (10 μm). H, Example of a whole-cell patch-clamp recording from an hTRPV1-expressing cell pretreated with ALA (300 μm, 3 h). The current induced by stimulation with blue light was inhibited by BCTC (1 μm). I, J, Ramp currents recorded at the times indicated with corresponding color in G and H. A–J, Carvacrol 100 μm (Carv.); and capsaicin 300 nm (Caps.).

ALA and PpIX photosensitize neuropeptide release by activation of TRPA1 and TRPV1. A, In isolated hindpaw skin, release of the neuropeptide CGRP was measured by EIA as index of neuronal activation. Compared with a period without illumination, an intense broad-spectrum light caused the release of CGRP from skin pretreated with PpIX (PpIX 5 μm for 40 min, n = 10–12). Compared with C57BL/6, CGRP release was substantially reduced in hindpaw skin of TRPA1−/− and TRPV1−/− animals and absent without pretreatment by PpIX (n = 8). B, Comparison of the light-induced increase in CGRP release after PpIX pretreatment in the different genotypes (n = 10–12, p < 0.05 vs C57BL/6). C, The isolated superfused mouse trachea was stimulated by 405 nm laser illumination after 1 h in control solution (SIF). A slight but significant increase in CGRP release was observed in trachea from WT animals (C57BL/6, n = 7); this was reduced in TRPA1−/− animals (n = 4) and in the presence of HC-030031 (n = 4) but not in TRPV1−/− animals (n = 4). D, After 1 h exposure to ALA (100 μm), stimulation by the 405 nm laser caused a substantial increase in CGRP release. Compared with WT mice (n = 6), the light-induced CGRP release was significantly reduced in the trachea of TRPA1−/− (n = 5), TRPV1−/− (n = 4), and TRPA1−/−/TRPV1−/− (n = 4) animals. Data in the trachea are given as increase of CGRP (ΔCGRP) over baseline.